1. Field of the Invention
This invention relates to antiviral peptide compounds and to methods of inhibiting infection of human cells by viruses. This invention pertains more specifically to peptides that are chemically blocked at the amino- and carboxy- termini. In particular the invention relates to peptides comprised of prolylalanine or prolylphenylalanine compounds that have antiviral activity. The invention is specifically directed to the inactivation of the human immunodeficiency virus and inhibition of infection of human cells in vivo and in vitro with this and other viruses. The invention also relates to the diagnostic and therapeutic use of these antiviral peptide compounds.
2. Background of the Related Art
The use of peptides having antiviral properties is known in the art. [See, Ringrose, Biochem. Soc. Trans. 11:804-808 (1983) for a review].
Miller et al., Applied Microbiol. 16: 1489-1496 (1967) describe the use of N-carbobenzoxy-derivatives of (D)-Phe-(D)-Phe, (L)-Phe-nitro-Arg, (D)-Phe-(D)-Phe-nitro-Arg, (D)-Phe-(D)-Met, (D)-Phe-Ala and (D)-Phe-S-benzyl-Cys to inhibit herpesvirus and measles virus infections in vivo and in vitro. These peptides were inactive against a wide variety of other viruses tested.
Mathur et al., Ind. J. Exp. Biol. 20:227-229 (1982) disclose the antiviral activity of poly(.alpha.-L-Lys) and poly(.epsilon.-L-Lys) against a number of double stranded RNA viruses in vivo, mediated by the induction of the antiviral protein interferon.
Konopinska et al., Int. J. Peptide Protein Res. 22: 223-230 (1983) disclose the antiviral activity of three tuftsin analogs (Thr-Lys-Pro-Lys-Thr-Lys-Pro-Lys, (Seq. ID. No.:1) Thr-Lys-Pro-Lys-Thr-Lys-Pro-Arg, (Seq. ID. No:2) and Ala-Lys-Thr-Lys-Pro-Arg-Gln-Gln) (Seq. ID. No:3) against murine sarcoma virus infection in vitro.
Pert et al., Proc. Natl. Acad. Sci. USA 83:9254-9258 (1986) disclose that the octapeptide Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr (Seq. ID. No:4) displays antiviral activity against human immunodeficiency virus (HIV-1) in vitro.
Dietrich et al., Int. J. Immunopharmac. 8: 931-942 (1986) teach the use of N-acetyl-muramyl-(L)-alanyl-(D)-isoglutaryl-(L)-alanine-2-(1',2'-dipalmito yl-sn-glycero-3'hydroxyphosphoryloxy)ethylamide sodium salt for prophylactic treatment of animals to prevent infection by influenza virus types A and B, parainfluenza virus 1 and herpes simplex virus types 1 and 2. This compound had no antiviral effect in vitro, however.
Daher et al., J. Virol. 60: 1068-1074 (1986) disclose the discovery of a naturally-occurring peptide of sequence ##STR1## derived from human neutrophil cells that inhibits infection of human cells by herpes simplex virus 1 and 2, cytomegalovirus, vesicular stomatitis virus and influenza virus A in vitro.
Docherty et al., Antimicrob. Agents and Chemother. 31: 1562-1566 (1987) teach the use of synthetic polymers of histidine (His.sub.24, His.sub.64, and His.sub.75) in vitro to effect the irreversible inhibition of infection of human cells with herpes simplex virus.
Lobl et al., Int. J. Protein Res. 32:326-330 (1988) teach the use of N-carbobenzoxy-(D)-Phe-Leu-Gly-(D)-Leu-(D)-Leu and N-carbobenzoxy-(D)-Phe-Leu-Gly-(D)-Leu-(D)-Leu-Gly to inhibit measles virus infection in vitro.
Naruse et al., J. Antibiotics 42:837-845 (1989) teach the use of a naturally-occurring peptide derived from Streptoverticillium cinnamoneum containing four unusual amino acids that displays antiviral activity against herpes simplex virus in vitro.
Srinivas et al., Virology 176:48-57 (1990) disclose the use of two synthetic peptides ##STR2## homologous to a region of the human apolipoprotein A-1 sequence, to inhibit infection and viral spread of herpes simplex virus 1 in vitro.
BJorck et al., J. Vir. 64: 941-943 (1990) teach that the synthetic peptide N-benzoxycarbonyl-leucylvalylglycine diazomethylketone (Z-LVG-CHN.sub.2) blocks the growth of herpes simplex virus but not poliovirus in vitro.
Inocencio et al., Med. Microbiol. Immunol. 179:87-94 (1990) disclose that the synthetic peptide Z-(D)-Phe-(L)-Phe displays antiviral activity against the paramyxoviruses rubeola, Sendal and Newcastle Disease virus in vitro.
Acquired immune deficiency syndrome (AIDS) is the most acute human public health problem to arise since the advent of the widespread use of antibiotics against bacterial infections over a generation ago. [See, Fauci, Science 239: 617-622 (1988) for a review]. The disease is widely believed to be caused by a virus, human immune deficiency virus 1 (HIV-1; also known as HTLV-III). The virus has been shown to enter human cells via its interaction with a specific cell surface receptor. This receptor, a glycoprotein termed CD4, is found on a specific class of human T lymphocytes that are the principle in vivo target for infection. This molecule is also a receptor for the Class II major histocompatibility complex (MHC) proteins that mediate immune recognition. Physiologically, CD4 is believed to bind a monomorphic domain on Class II MHC, thereby facilitating antigen recognition and enhancing T cell activation. Loss of this subset of T lymphocytes as a result of infection with HIV-1 results in the immune deficiency disorder. In addition, binding of CD4 by gp120 blocks the ability of CD4 to bind to Class II MHC or to be stimulated by Class II MHC, thereby interfering with effective immunological response to infection by HIV-1.
One approach to developing a method for preventing human infection with HIV-1 is to attempt to prevent binding between CD4 and HIV-1. Initial efforts involved the use of soluble forms of CD4, produced by expression of truncated forms of the protein by genetic engineering means. However, the use of the soluble forms of CD4 as therapeutic agents presents several problems in terms of delivery, stability and expense.
Smith et al., Science 238: 1704-1707 (1987) disclose the use of a soluble form of CD4, produced in CHO cells by expression and secretion of a truncated form of the protein directed by a transfected copy of a cDNA clone of the receptor gene, to inhibit HIV-1 infection of CD4.sup.+ human cells in vitro.
Fischer, et al., Nature 331:76-78 (1988) disclose the use of a soluble form of CD4, produced in CHO cells by expression and secretion of a truncated form of the protein directed by a transfected copy of a cDNA clone of the receptor gene, to inhibit HIV-1 infection of CD4.sup.+ human cells in vitro.
Hussey et al. Nature 331: 78-81 (1988) disclose the use of a soluble form of CD4, produced in insect cells transfected with a baculovirus vector containing a truncated CD4 cDNA, to inhibit HIV-1 infection of CD4.sup.+ human cells in vitro.
Deen et al., Nature 331: 82-84 (1988) disclose the use of a soluble form of CD4, produced in CHO cells by expression and secretion of a truncated form of the protein directed by a transfected copy of a cDNA clone of the receptor gene, to inhibit HIV-1 infection of CD4.sup.+ human cells in vitro.
Traunecker et al., Nature 331: 84-86 (1988) disclose the use of a soluble form of CD4, produced in myeloma cells by expression and secretion of a truncated form of the protein directed by a transfected copy of a cDNA clone of the receptor gene, to inhibit HIV-1 infection of CD4.sup.+ human cells in vitro.
It is known that the envelope glycoprotein of the virus, gp120, binds to CD4 by way of specific binding sites present in both molecules that mediate their recognition. The respective binding sites of both glycoproteins have been mapped. Subsequent efforts have focused on the use of CD4-derived synthetic peptides to inhibit binding of CD4 to HIV-1 gp120.
Jameson et al., Science 240: 1335-1339 (1988) disclose the use of a synthetic peptide, ##STR3## derived from the deduced CD4-gp120 binding site by immunological studies, to bring about a decrease in HIV-1 induced cell fusion in vitro.
Lifson et al., Science 241:712-716 (1988) disclose the use of a mixture of the synthetic peptide ##STR4## derived from CD4, and side products produced during the synthesis of this peptide, that inhibit HIV-1 infection and cytopathicity in vitro.
Nara et al., Proc. Natl. Acad. Sci. USA 86:7139-7143 (1989) teach the use of the synthetic peptide ##STR5## derived from CD4, to inhibit HIV-1 infection in vitro.
Thus, as summarized herein, it was known in the prior art that peptides derived from epitopes of CD4 that had an inhibitory effect on HIV-1 infection also prevented the binding of epitope-specific anti-CD4 antibodies. It was also known in the art that the efficiency of inhibition of virus infection increased with the size of the peptide (Jameson et al., supra).
Bowman et al., Proc. Natl. Acad. Sci. USA 87:9052-9056 (1990) examined the binding interaction between CD4 and gp120 using epitope loss mutants, produced by genetic engineering means and expressed in otherwise CD4.sup.- cells in vitro, and found that a particular class of such mutants specifically resulted in loss of the ability to bind gp120. These mutants were unique in that they did not display any change in their ability to recognize epitope-specific anti-CD4 antibodies. This result indicated that the alteration in the gp120 binding ability of these mutants was not the result of dramatic changes in the conformation of the CD4 protein, and suggested that this particular epitope might specifically interact with gp120. Molecular modeling studies showed that this peptide had predominantly a .beta.-sheet secondary structure, and that in this structure the sidechain of Phe.sup.43 would conspicuously protrude from the surface of the protein.
Finberg et al., Science 249: 287-291 (1990) designed and tested a number of amino- and carboxy-terminally blocked di- and tri-peptides for their ability to inhibit binding of HIV-1 gp120 to CD4 expressed on HBS cells. Some of the peptides tested displayed the inhibitory activity and are among those comprehended by the present invention. Antiviral peptides according to the present invention are unique in that these peptides specifically bind to gp120 rather than to CD4.